The long term goal of this project is to contribute to understanding how structures and components involved in cell reproduction are regulated by the cell cycle engine. This research is based on the use of MPM-2, a monoclonal antibody recognizing a phosphorylated epitope found on a large number of proteins during M phase, which are putative targets of cell cycle regulation. In the previous project period, cDNA clones encoding portions of 11 novel MPM-2 antigens were isolated. In this period, detailed analysis is planned for three of these proteins. These antigens are localized to the centrosome (MPP3), DNA replication centers (MPP7) and the Golgi apparatus (MPP9), which are important regulatory targets of the cell cycle machinery in interphase and/or mitosis. cDNA clones yielding full length coding sequences of MPPs3, 7 and 9 will be isolated and sequenced, and used to prepare antibodies for biochemical, localization, and functional studies. Phosphorylation and expression of MPPs3, 7 and 9 through the cell cycle will be analyzed by metabolic labeling and peptide mapping, and cdk-cyclin complex(es) responsible for phosphorylation of these components at discrete periods of the cell cycle will be analyzed with in vitro and in vivo studies. Functions of MPPs3, 7 and 9 in the centrosome, DNA replication centers and Golgi will be investigated by in vivo approaches including microinjection of antibodies and antisense oligonucleotides, and expression of antisense RNA, ribozymes and putative dominant negative mutants in cultured cells. In vitro functional studies will include cell-free assays for microtubule nucleation by the centrosome, DNA replication and membrane trafficking. Finally, interaction partners of these proteins will be determined by biochemical methods and a yeast two- hybrid system to provide a basis for further functional understanding. Together, these studies are expected to provide important insight on how some major features of cell organization are regulated by the cyclin-cdk system.